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Microbes & Immunity Establishment of a novel anti-human CCR8 monoclonal antibody

these molecules could potentiate the activation and 2. Materials and methods
immunosuppressive function of Treg. 10-12
2.1. Cell lines
Treg is defined as CD4 T cell that expresses CD25
+
and FOXP3, playing a role in maintaining self-tolerance LN229, Chinese hamster ovary (CHO)-K1, and
to prevent excessive immune responses and autoimmune P3X63Ag8U.1 (P3U1) cells were obtained from the
diseases. 13,14 Treg suppresses the effector functions of American Type Culture Collection (Manassas, VA, USA).
T cells through the secretion of immunosuppressive TALL-1 and CCRF-HSB2 cells were obtained from the
cytokines, such as interleukin-10 (IL-10), transforming Japanese Collection of Research Bioresources Cell Bank
growth factor-β, and cytotoxic granzyme/perforin. 14-18 (Osaka, Japan). The human natural killer (NK) cells
Intratumoral Treg suppresses antitumor T cell responses (donor lot. 4022602, purity >70%) were purchased from
and thus resists the effects of immune checkpoint inhibitor Takara Bio (Shiga, Japan). pCMV6neo-myc-DDK vector
therapy. 19,20 Antibodies against T cell immunoreceptors with hCCR8 (Accession No.: NM_005201) was purchased
with Ig and ITIM domains (TIGIT), one of the immune from OriGene Technologies, Inc. (Rockville, MD, USA).
checkpoint molecules, have shown to improve the The plasmid was transfected into cell lines using the
effectiveness of PD-L1 antibodies by suppressing Treg. Neon transfection system (Thermo Fisher Scientific, Inc.,
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Therefore, the development of immunotherapy targeting Waltham, MA, USA). Subsequently, LN229 and CHO-K1
Treg is expected. 11,22 stably overexpressing hCCR8 with C-terminal myc-DDK
tags (hereinafter described as LN229/hCCR8 and CHO/
Intratumoral Treg expresses high levels of C-C hCCR8, respectively) were established using a cell sorter
4
motif chemokine receptor-8 (CCR8). In addition, (SH800; Sony Corp., Tokyo, Japan), following cultivation
CCR8-expressing Tregs exhibit increased expression of in a medium containing 0.5 mg/mL G418 (Nacalai Tesque,
CD25 and FOXP3 compared to CCR8-negative Tregs, Inc., Kyoto, Japan).
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indicating their potent immunosuppressive functions.
The CCR8-expressing Treg is known to be correlated with CHO-K1, P3U1, CHO/hCCR8, TALL-1, and CCRF-
poor prognosis in some cancer patients. 24,25 Thus, CCR8 HSB2 cells were cultured in a Roswell Park Memorial
is emerging as an attractive target for the next cancer Institute (RPMI)-1640 medium (Nacalai Tesque, Inc.,
immunotherapy. Several anti-CCR8 drugs, including Kyoto, Japan) supplemented with 10% heat-inactivated
26
S-531011, IPG7236, and SRF114 are undergoing fetal bovine serum (FBS, Thermo Fisher Scientific
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27
28
clinical trials. Inc., Waltham, MA, USA), 100 units/mL penicillin,
100 μg/mL streptomycin, and 0.25 μg/mL amphotericin
CCR8 is one of the seven transmembrane-spanning B (Nacalai Tesque, Inc., Kyoto, Japan). LN229 and
G protein-coupled receptors. Human CCR8 (hCCR8) LN229/hCCR8 were cultured in a Dulbecco’s Modified
4
is known to bind to five C-C chemokine ligands (CCLs): Eagle Medium (DMEM, Nacalai Tesque, Inc., Kyoto,
CCL1/I-309, CCL4, CCL16, CCL17, and CCL18. 30-32 CCR8 Japan) supplemented with 10% heat-inactivated FBS
is upregulated not only in Treg but also in various cancers, (Thermo Fisher Scientific Inc., Waltham, MA, USA),
including breast, non-small cell lung (NSCLC), bladder, 100 units/mL penicillin, 100 μg/mL streptomycin, and
and colorectal cancer. 24,25 In bladder cancer, CCR8 mediates 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc., Kyoto,
cell migration, invasion, and epithelial-mesenchymal Japan). All cells were cultured in a humidified incubator
transition by interacting with CCL18. In addition, CCR8 at 37°C with 5% CO and 95% air.
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and its specific ligand CCL1/I-309 regulate the immune 2
system, which mediates the progression of diseases such as 2.2. Antibodies
cancers by promoting migration and inhibiting apoptosis The anti-human CD198 (CCR8) mAb (clones S19017D
in Treg and lymphomas. 34,35 Therefore, CCR8-targeting and L263G8) were purchased from BioLegend (San Diego,
antibodies will contribute to the elucidation of pathological CA, USA). The Alexa Fluor 488-conjugated anti-mouse
mechanisms, diagnosis, and therapy. 27,36 Immunoglobulin (Ig)G was purchased from Cell Signaling

Using the Cell-Based Immunization and Screening Technology, Inc. (Danvers, MA, USA).
(CBIS) method, we previously developed numerous 2.3. Hybridoma production
monoclonal antibodies (mAbs) against chemokine
receptors, including mouse CCR3, mouse CCR8, For developing anti-hCCR8 mAbs, two female 6-week-old
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37
human CCR9, and mouse C-X-C chemokine receptor BALB/c mice were immunized intraperitoneally with 1 ×
39
type 4 (CXCR4). In this study, we successfully developed 10 cells of LN229/hCCR8. The immunogen was harvested
40
8
an anti-hCCR8 mAb using the CBIS method, which is after brief exposure to 1 mM ethylenediaminetetraacetic
applicable to flow cytometry. acid (EDTA; Nacalai Tesque, Inc., Kyoto, Japan). Imject
Volume 2 Issue 2 (2025) 127 doi: 10.36922/mi.4661